U.S. patent number 10,517,854 [Application Number 15/706,845] was granted by the patent office on 2019-12-31 for dosing regiments of celgosivir for the treatment of dengue.
This patent grant is currently assigned to 60 Degrees Pharmaceuticals LLC, National University of Singapore, Singapore Health Services PTE Ltd.. The grantee listed for this patent is 60 Degrees Pharmaceuticals, LLC, National University of Singapore, Singapore Health Services PTE Ltd.. Invention is credited to Geoffrey S. Dow, Jenny Low, Glynn Morrish, Eng Eong Ooi, Abhay Rathore, Mark Reid, Cynthia Sung, Subhash Vasudevan, Satoru Watanabe.
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United States Patent |
10,517,854 |
Dow , et al. |
December 31, 2019 |
Dosing regiments of celgosivir for the treatment of dengue
Abstract
Methods of treating a dengue virus (DENV) infection in a human
subject, comprising administering to the human subject a compound
of Formula (I), or pharmaceutical composition comprising a compound
of Formula (I): A compound of Formula (I) can be administered
within onset of fever to 72 hours of fever onset due to dengue
infection and then every 6 to 12 hours until there is an
improvement in the infection or between from about 1 day to about
10 days. The methods of the invention can be used to treat primary
and secondary DENV 1-4 viral infections.
Inventors: |
Dow; Geoffrey S. (Washington,
DC), Vasudevan; Subhash (Singapore, SG), Reid;
Mark (Toowong, AU), Morrish; Glynn (Toowong,
AU), Sung; Cynthia (Singapore, SG),
Rathore; Abhay (Singapore, SG), Watanabe; Satoru
(Singapore, SG), Ooi; Eng Eong (Singapore,
SG), Low; Jenny (Singapore, SG) |
Applicant: |
Name |
City |
State |
Country |
Type |
60 Degrees Pharmaceuticals, LLC
National University of Singapore
Singapore Health Services PTE Ltd. |
Washington
Singapore
Singapore |
DC
N/A
N/A |
US
SG
SG |
|
|
Assignee: |
60 Degrees Pharmaceuticals LLC
(Washington, DC)
National University of Singapore (Singapore, SG)
Singapore Health Services PTE Ltd. (Singapore,
SG)
|
Family
ID: |
51537558 |
Appl.
No.: |
15/706,845 |
Filed: |
September 18, 2017 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20180064693 A1 |
Mar 8, 2018 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14772873 |
Sep 19, 2017 |
9763921 |
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PCT/US2014/028076 |
Mar 14, 2014 |
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61911795 |
Dec 4, 2013 |
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61798100 |
Mar 15, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
31/14 (20180101); A61K 31/437 (20130101); Y02A
50/30 (20180101); Y02A 50/385 (20180101) |
Current International
Class: |
A61K
31/437 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 01/54692 |
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Aug 2001 |
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WO |
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WO 02/089780 |
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Nov 2002 |
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WO |
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Other References
International Search Report dated Jul. 29, 2014, in corresponding
PCT Application No. PCT/US2014/028076. cited by applicant .
Low et al., Celgosivir as a Treatment Against Dengue,
clinicaltrials.gov Identifier: NCT01619969 (Oct. 19, 2012). cited
by applicant .
PubChem Compound Summary for : CID 60734, Celgosivir,
https://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=60734>
(Jun. 26, 2014). cited by applicant .
Watanabe et al., Dose- and schedule-dependent protective efficacy
of celgosivir in a lethal mouse model for dengue virus infection
informs dosing regimen for a proof of concept clinical trial, 96
Antiviral Research 32-35 (2012). cited by applicant .
Search Report and Written Opinion dated Oct. 26, 2016, in
corresponding Singapore Patent Application No. 11201507254V. cited
by applicant .
Rathore, et al., Celgosivir treatment misfolds dengue virus NS1
protein, induces cellular pro-survival genes and protects against
lethal challenge mouse model, 92 Antiviral Research 453-460 (2011).
cited by applicant .
Chaterji et al., Evaluation of the NS1 Rapid Test and the WHO
Dengue Classification Schemes for Use as Bedsides Diagnosis of
Acute Dengue Fever in Adults, 84(2) Am. J. Trop. Med. 224-228
(2011). cited by applicant.
|
Primary Examiner: Anderson; James D.
Attorney, Agent or Firm: Buchanan Ingersoll & Rooney
PC
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application a Continuation Application of U.S. patent
application Ser. No. 14/772,873, filed on Sep. 4, 2015, now U.S.
Pat. No. 9,763,921, issued on Sep. 19, 2017, which is a U.S.
National Phase Application pursuant to 35 U.S.C. .sctn. 371 of
International Patent Application No. PCT/US2014/028076, filed on
Mar. 14, 2014, and published as WO 2014/143907 on Sep. 18, 2014,
which claims the benefit of U.S. Provisional Patent Application No.
61/798,100, filed on Mar. 15, 2013, and U.S. Provisional Patent
Application No. 61/911,795, filed on Dec. 4, 2013, the entireties
of which are incorporated herein by reference for all purposes.
Claims
What is claimed is:
1. A method of treating a dengue virus (DENV) infection in a human
subject, comprising: a) administering to the human subject an
initial dose of about 100 to about 400 mg of a compound of Formula
(II), or a pharmaceutical composition comprising a compound of
Formula (II), within from about onset of fever to about 72 hours of
fever onset due to dengue infection; and b) administering to the
human subject a dose of about 150 to about 400 mg of a compound of
Formula (II), or a pharmaceutical composition comprising a compound
of Formula (II), at intervals of about 24 hours until there is an
improvement in the infection or between from about 1 day to about
10 days, Formula (II) having the following structure, ##STR00007##
or a pharmaceutically acceptable salt thereof.
2. The method of claim 1, wherein dengue viral infection comprises
at least one dengue virus selected from DENV1, DENV2, DENV3 and
DENV4.
3. The method of claim 1, wherein the dengue viral infection is
secondary dengue infection.
4. The method of claim 1, wherein the compound, or the
pharmaceutical composition, is administered intravenously, orally,
rectally, or sublingually.
5. The method of claim 1, wherein the human subject is administered
an initial dose of 300 mg and a dose of 300 mg is thereafter
administered to the human subject about every 24 hours for about 1
day, about 2 days, or about 5 days.
6. The method of claim 1, wherein the human subject is administered
an initial dose of 150 mg and a dose of 150 mg is thereafter
administered to the human subject about every 24 hours for about 1
day, about 2 days, or about 5 days.
7. The method of claim 1, wherein the human subject is administered
an initial dose of 400 mg and a dose of 400 mg is thereafter
administered to the human subject about every 24 hours for about 1
day, about 2 days, or about 5 days.
8. The method of claim 1, wherein the human subject is administered
an initial dose of 300 mg and a dose of 150 mg is thereafter
administered to the human subject about every 24 hours for about 1
day, about 2 days, or about 5 days.
9. The method of claim 1, wherein the human subject is administered
an initial dose of 150 mg and a dose of 200 mg is thereafter
administered to the human subject about every 24 hours for about 1
day, about 2 days, or about 5 days.
10. The method of claim 1, wherein the human subject is
administered an initial dose of 200 mg and a dose of 300 mg is
thereafter administered to the human subject about every 24 hours
for about 1 day, about 2 days, or about 5 days.
11. The method of claim 1, wherein the human subject is
administered an initial dose of 200 mg and a dose of 150 mg is
thereafter administered to the human subject about every 24 hours
for about 1 day, about 2 days, or about 5 days.
12. The method of claim 1, wherein the human subject is
administered an initial dose of 200 mg and a dose of 200 mg is
thereafter administered to the human subject about every 24 hours
for about 1 day, about 2 days, or about 5 days.
13. The method of claim 1, wherein the human subject is
administered an initial dose of 150 mg and a dose of 300 mg is
thereafter administered to the human subject about every 24 hours
for about 1 day, about 2 days, or about 5 days.
14. The method of claim 1, wherein the human subject is
administered an initial dose of 150 mg and a dose of 400 mg is
thereafter administered to the human subject about every 24 hours
for about 1 day, about 2 days, or about 5 days.
15. The method of claim 1, wherein the human subject is
administered an initial dose of 200 mg and a dose of 400 mg is
thereafter administered to the human subject about every 24 hours
for about 1 day, about 2 days, or about 5 days.
16. The method of claim 1, wherein the human subject is
administered an initial dose of 300 mg and a dose of 200 mg is
thereafter administered to the human subject about every 24 hours
for about 1 day, about 2 days, or about 5 days.
17. The method of claim 1, wherein the human subject is
administered an initial dose of 300 mg and a dose of 400 mg is
thereafter administered to the human subject about every 24 hours
for about 1 day, about 2 days, or about 5 days.
18. The method of claim 1, wherein the human subject is
administered an initial dose of 400 mg and a dose of 150 mg is
thereafter administered to the human subject about every 24 hours
for about 1 day, about 2 days, or about 5 days.
19. The method of claim 1, wherein the human subject is
administered an initial dose of 400 mg and a dose of 200 mg is
thereafter administered to the human subject about every 24 hours
for about 1 day, about 2 days, or about 5 days.
20. The method of claim 1, wherein the human subject is
administered an initial dose of 400 mg and a dose of 300 mg is
thereafter administered to the human subject about every 24 hours
for about 1 day, about 2 days, or about 5 days.
Description
BACKGROUND OF THE INVENTION
Globally, dengue infections result in more than 20,000 deaths,
nearly 500,000 hospitalized cases and anywhere between 50-100
million human infections annually. (1) Dengue infection is caused
by one of four immunologically distinct serotypes of the dengue
virus (DENV 1-4). The virus is spread by the urban breeding
mosquito Aedes aegypti. Usually, infection with any one of the four
DENV serotypes leads to mild, self-limiting dengue fever with
lifelong immunity to the specific serotype of infection.
Epidemiological evidence also indicates that 90% of the severe and
potentially fatal dengue diseases, dengue hemorrhagic fever (DHF)
or dengue shock syndrome (DSS) occur during secondary heterotypic
infections where the protective antibody from a prior infection
takes on a pathogenic role, so-called Antibody Dependent
Enhancement (ADE). (2, 3) The antibody response triggers a systemic
inflammatory reaction resulting in vascular leakage.
Currently, there is no approved preventive vaccine or antiviral
treatment for dengue disease. The World Health Organization has
listed dengue fever as an emerging and uncontrolled disease. (4)
Hence, there is an urgent medical need for the development of a
potent dengue antiviral that is safe for use in humans.
SUMMARY OF THE INVENTION
The present invention pertains to methods of treating a dengue
virus (DENV) infection in a human subject. In one aspect, the
method comprises administering to the human subject an initial
(loading) dose of about 100 to 600 mg of a compound of Formula (I),
or a pharmaceutical composition comprising a compound of Formula
(I), within from about onset of fever to about 72 hours of fever
onset due to dengue infection, followed by administration of one or
more subsequent doses of about 25 to about 300 mg of a compound of
Formula (I), or a pharmaceutical composition comprising a compound
of Formula (I).
In one embodiment, the subsequent doses are administered at
intervals of from about 6 to about 12 hours. In other embodiments,
the subsequent doses are administered about every 6 hours, about
every 8 hours, about every 12 hours, or about every 24 hours.
In certain embodiments, the subsequent doses are administered for
about 1-10 days. In other embodiments, the subsequent doses are
administered for about 1-5 days, 1-4 days, 1-3 days, 2-3 days, or
1-2 days. In further embodiments, the subsequent doses are
administered until there is an improvement in the infection or its
symptoms.
In the various embodiments, the human subject can be an adult or a
child.
The compound of Formula (I) is represented by the following
structure:
##STR00001## or a pharmaceutically acceptable salt thereof;
wherein R.sup.1, R.sup.2, and R.sup.3 are independently H,
(C.sub.1-C.sub.14) acyl, (C.sub.1-C.sub.14) alkenylacyl,
(C.sub.3-C.sub.8) cycloalkylacyl, (C.sub.1-C.sub.14) haloalkylacyl
(C.sub.1-C.sub.8) alkoxyacyl, or (C.sub.6-C.sub.10) arylacyl.
In certain embodiments, the compound of Formula (I) is specifically
the compound of Formula (II), below, or a pharmaceutically
acceptable salt thereof:
##STR00002##
In preferred embodiments, the compound of Formula (I) is prodrug of
castanospermine, a natural product derived from the seeds of
Castanospermum austral. Once administered compounds of Formula (I)
are rapidly converted to castanospermine. Compounds of Formula (I)
(e.g., celgosivir) are more rapidly and efficiently absorbed than
castanospermine. Compounds of Formula (I) are also more readily
absorbed into cells. As a result, compounds of Formula (I) may have
higher 50% effective concentration (EC50) values and in vivo
efficacy than castanospermine against the dengue (DENV) virus.
In certain embodiments, the initial dose is the same as the
subsequent doses, while in other embodiments the initial dose
differs from the subsequent doses. In particular embodiments, the
initial dose is higher than the subsequent doses.
In certain embodiments, for an adult subject, the initial dose of a
compound of Formula (I) can be between about 100 to about 600 mg.
In other embodiments, the initial dose of a compound of Formula (I)
in an adult subject can be about 150-600 mg, about 200-500 mg, or
about 250-400 mg. In further embodiments, the initial dose of a
compound of Formula (I) in an adult subject can be about 100 mg,
about 125 mg, about 150 mg, about 175 mg about 200 mg, about 225
mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about
350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg,
about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575
mg, or about 600 mg. In a further embodiment, the initial dose of a
compound of Formula (I) in an adult subject is between about 550 to
about 600 mg. In another embodiment, the initial dose of a compound
of Formula (I) in an adult subject is between about 500 to about
550 mg. In yet another embodiment, the initial dose of a compound
of Formula (I) in an adult subject is between about 450 to about
500 mg. In further embodiment, the initial dose of a compound of
Formula (I) in an adult subject is between about 400 to about 450
mg. In another embodiment, the initial dose of a compound of
Formula (I) in an adult subject is between about 350 to about 400
mg. In further embodiment, the initial dose of compound of Formula
(I) in an adult subject is between about 300 to about 350 mg. In
yet another embodiment, the initial dose of a compound of Formula
(I) in an adult subject is between about 250 to about 300 mg. In
further embodiment, the initial dose of compound of Formula (I) in
an adult subject is between about 200 to about 250 mg. In another
embodiment, the initial dose of a compound of Formula (I) in an
adult subject is between about 150 to about 200 mg. In another
embodiment, the initial dose of compound of Formula (I) in an adult
subject is between about 100 to about 150 mg.
The subsequent doses of a compound of Formula (I) in an adult
subject can be between about 100 to about 300 mg. In one
embodiment, the subsequent dose of a compound of Formula (I) in an
adult subject is between about 250 to about 300 mg. In another
embodiment the subsequent dose of a compound of Formula (I) in an
adult subject is between about 200 to about 250 mg. In yet another
embodiment, the subsequent dose of a compound of Formula (I) in an
adult subject is between about 150 to about 200 mg. In further
embodiment, the subsequent dose of a compound of Formula (I) in an
adult subject is between about 100 to about 200 mg. In an
additional embodiment, the subsequent dose of a compound of Formula
(I) in an adult subject is between about 125 to about 175 mg. In
yet another embodiment, the subsequent dose of a compound of
Formula (I) in an adult subject is about 150 mg.
For a child subject, the initial dose of a compound of Formula (I)
in a child subject can be between about 25 to about 450 mg. In one
embodiment, the initial dose of a compound of Formula (I) in a
child subject is between about 25 to about 50 mg. In another
embodiment, the initial dose of a compound of Formula (I) in a
child subject is between about 50 to about 75 mg. In yet another
embodiment, the initial dose of a compound of Formula (I) in a
child subject is between about 75 to about 100 mg. In further
embodiment, the initial dose of a compound of Formula (I) in a
child subject is between about 100 to about 150 mg. In another
embodiment, the initial dose of a compound of Formula (I) in a
child subject is between about 150 to about 200 mg. In yet another
embodiment, the initial dose of a compound of Formula (I) in a
child subject is between about 200 to about 250 mg. In further
embodiment, the initial dose of a compound of Formula (I) in a
child subject is between about 250 to about 300 mg. In another
embodiment, the initial (lose of a compound of Formula (I) in a
child subject is between about 300 to about 350 mg. In yet another
embodiment, the initial dose of a compound of Formula (I) in a
child subject is between about 350 to about 400 mg.
The subsequent doses of a compound of Formula (I) in a child
subject can be between about 25 to about 200 mg. In one embodiment,
the subsequent dose of a compound of Formula (I) in a child subject
is between about 25 to about 50 mg. In another embodiment, the
subsequent dose of a compound of Formula (I) in a child subject is
between about 50 to about 75 mg. In yet another embodiment, the
subsequent dose of a compound of Formula (I) in a child subject is
between about 75 to about 100 mg. In further embodiment, the
subsequent dose of a compound of Formula (I) in a child subject is
between about 100 to about 125 mg. In another embodiment, the
subsequent dose of a compound of Formula (I) in a child subject is
between about 125 to about 150 mg. In yet another embodiment, the
subsequent dose of a compound of Formula (I) in a child subject is
between about 150 to about 200 mg.
In one embodiment, the initial dose is administered at the time of
lever onset due to dengue infection. In another embodiment, the
initial dose is administered within 24 hours of fever onset due to
dengue infection. In yet another embodiment, the initial dose is
administered within 48 hours of fever onset due to dengue
infection. In a further another embodiment, the initial dose is
administered within 72 hours of fever onset due to dengue
infection.
The compounds or pharmaceutical compositions of the present
invention can be administered intravenously, orally, rectally or
sublingually. In one embodiment, the route of administration is
intravenous. In another embodiment, the route of administration is
oral. In another embodiment, the route of administration is rectal.
In yet another embodiment, the route of administration is
sublingual.
The compounds or pharmaceutical compositions of the present
invention can be administered as a single or as a divided dose.
Preferably, the initial dose is a single dose. In some embodiments,
the subsequent doses can be single, divided, or a combination
thereof, during the course of therapy depending upon patient and
progress of the infection. For the subsequent doses in one
embodiment, the human subject is administered a divided dose of
from about 25 to about 300 mg of a compound of Formula (I), or a
pharmaceutical composition comprising a compound of Formula (I),
for between about 1 day to about 10 days. In another embodiment,
the human subject is administered a single dose of from about 25 to
about 300 mg of a compound of Formula (I), or a pharmaceutical
composition comprising a compound of Formula (I), for between about
1 day to about 10 days. In yet another embodiment, the human
subject is administered a single dose of from about 25 to about 300
mg of a compound of Formula (I), or a pharmaceutical composition
comprising a compound of Formula (I), for between about 1 day to
about 2 days. In a further embodiment, the human subject is
administered a single dose of from about 25 to about 300 mg of a
compound of Formula (I), or a pharmaceutical composition comprising
a compound of Formula (I), for between about 2 days to about 5
days. In other versions, the subsequent doses are administered no
longer than about 1 day; in yet other versions no longer than about
2 days; in further versions no longer than about 5 days; and other
versions no longer than about 10 days.
The invention also relates to methods of treating a dengue virus
infection by achieving a steady state Cmin serum or plasma
concentration of between about 0.05 and about 2.0 microgram/mL of
castanospermine in an adult or child subject. In one embodiment,
the steady state Cmin serum or plasma concentration achieved in an
adult or child subject is between about 0.08 and about 0.5
microgram/mL of castanospermine. In another embodiment, the steady
state Cmin serum or plasma concentration achieved in an adult or
child subject is between about 0.05 and about 0.08 microgram/mL of
castanospermine. In yet another embodiment, the steady state Cmin
serum or plasma concentration achieved in an adult or child subject
is between about 0.08 and about 0.11 microgram/mL of
castanospermine. In a further embodiment, the steady state Cmin
serum or plasma concentration achieved in an adult or child subject
is between about 0.11 and about 0.3 microgram/mL of
castanospermine. In another embodiment, the steady state Cmin serum
or plasma concentration achieved in an adult or child subject is
between about 0.3 and about 0.75 microgram/mL of castanospermine.
In further embodiment, the steady state Cmin serum or plasma
concentration achieved in an adult or child subject is between
about 0.75 and about 1.0 microgram/mL of castanospermine. In yet
another embodiment, the steady state Cmin serum or plasma
concentration achieved in an adult or child subject is between
about 1.0 and about 2.0 microgram/mL of castanospermine. In another
embodiment, the steady state Cmin serum or plasma concentration
achieved in an adult or child subject is between about 1.0 and
about 1.5 microgram/mL of castanospermine. In further embodiment,
the steady state Cmin serum or plasma concentration achieved in an
adult or child subject is between about 1.5 and about 2.0
microgram/mL of castanospermine. In yet another embodiment, the
steady state Cmin serum or plasma concentration achieved in an
adult or child subject is between about 1.25 and about 1.75
microgram/mL of castanospermine.
In another aspect, the invention relates to methods for treating a
dengue viral infection comprising at least one dengue virus
selected from DENV1, DENV2, DENV3 and DENV4. In one embodiment, the
dengue virus is DENV1. In another embodiment, the dengue virus is
DENV2. In yet another embodiment, the dengue virus is DENV3. In
further embodiment, the dengue virus is DENV4.
In yet another aspect, the invention relates to methods of treating
a dengue viral infection in a human subject who has tested positive
for dengue virus. Known methods for diagnosis of dengue viral
infection can be used including, but are not limited to, an NS1
(nonstructural protein 1) strip assay or a quantitative Polymerase
Chain Reaction (PCR) assay. The selected method should be rapid
enough for a diagnosis within from about onset of fever to about 72
hours of fever onset to optimize the therapeutic regimen of the
various embodiments of the invention.
The invention also relates to methods of treating a secondary
dengue (DENV) viral infection in a human subject, comprising
administering to the human subject an initial dose of about 100 to
about 600 mg of a compound of Formula (I), or a pharmaceutical
composition comprising a compound of Formula (I), within from about
onset of lever to about 72 hours of fever onset due to dengue
infection and administering to the human subject a dose of about 25
to about 300 mg of a compound of Formula (I), or a pharmaceutical
composition comprising a compound of Formula (I), at intervals of
from about 6 to about 12 hours until there is an improvement in the
infection or between from about 1 day to about 10 days.
In another aspect of the invention, viral load reduction of treated
human subjects is at least 50% greater than untreated or
placebo-treated human subjects. In one embodiment, the virological
log reduction in human subjects treated with a compound of Formula
(I) is at least 50% greater than untreated or placebo-treated
groups. In another embodiment, the virological log reduction in
human subjects treated with a compound of Formula (I) is between
about 60 to about 70% greater than untreated or placebo-treated
groups. In another embodiment, the virological log reduction in
human subjects treated with a compound of Formula (I) is between
about 70 to about 80% greater than untreated or placebo-treated
groups. In yet another embodiment, the virological log reduction in
human subjects treated with a compound of Formula (I) is between
about 80 to about 90% greater than untreated or placebo-treated
groups.
The invention is also directed to methods of treating a dengue
viral infection comprising administering a pharmaceutical
composition comprising a compound of Formula (I), Formula (II), or
Formula (III) according to any one of the dosing regimens described
herein. The disclosed compounds of Formula (I), Formula (II) or
Formula (III) can be administered to the subject in conjunction
with an acceptable pharmaceutical carrier or diluent as part of a
pharmaceutical composition for treatment of a dengue (DENV)
infection.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing will be apparent from the following more particular
description of example embodiments of the invention, as illustrated
in the accompanying drawings.
FIG. 1 shows a concentration response curve of celgosivir on DENV2
infection in the Cell-based flaviviral immunodetection assay (CFI)
assay; EC50 ranges from 0.2 to 0.7 .mu.M.
FIG. 2 shows immunofluorescence microscopy of untreated and
celgosivir (20 .mu.M) treated DENV2 infected BHK21. Nuclei are
stained with blue fluorescent DAPI. Virus replication was detected
using monoclonal anti-NS1 antibody (green fluorescence). The top
row shows that untreated, infected cells have abundant NS1 in the
cytoplasm while the bottom row shows that infected cells treated
with celgosivir have very low levels of NS1. Low levels of NS1
indicate suppression of viral replication.
FIG. 3 shows the concentration response curve of celgosivir on
DENV2 infection in the replicon assay (EC.sub.50 =2.2 .mu.M).
FIG. 4A shows the ADE effect-antibody against dengue E protein
increases viremia in THP-1 human monocytes infected with dengue
virus. FIG. 4B shows the effect of celgosivir on DENV2 infected
human monocytes pretreated with antibody to DENV E protein.
FIG. 5A shows a schematic of the dosing conducted in the DENV2
infected mice in a lethal ADE model of viremia. FIG. 5B shows
celgosivir improves survival of DENV2 infected mice in a lethal ADE
model of viremia in a dose- and schedule-dependent manner. Survival
at day 12 was 1/8 (13%) at 10 mg/kg twice daily (BID), 5/8 (63%) at
25 mg/kg BID, 7/7 (100%) at 50 mg/kg BID, and 0/8 (0%) at 100 mg/kg
once daily (QD).
FIG. 6A shows a concentration profile of celgosivir and
castanospermine after a single 50 mg/kg IP dose. FIG. 6B shows a
castanospermine profile calculated for 50 mg/kg BID dosing
regimen.
FIG. 7 shows the castanospermine concentration-time data over the
treatment period. Circles represent oberved concentrations. Shaded
grey area represents the model predicted 10.sup.th to 90.sup.th
prediction interval. Solid line represents the 50.sup.th prediction
interval.
FIG. 8A shows a simulated population mean castanospermine exposure
after a 400 mg loading dose of celgosivir followed by 9 subsequent
doses of 200 mg of celgosivir given at 12 hour intervals. 8B shows
a simulated population mean castanospermine exposure after a 400 mg
loading dose of celgosivir followed by 4 subsequent doses of 200 mg
of celgosivir given at 12 hour intervals. 8C shows a simulated
population mean castanospermine exposure after a 400 mg loading
dose of celgosivir followed by 8 subsequent doses of 100 mg of
celgosivir given at 6 hour intervals. 8D shows a simulated
population mean castanospermine exposure after a 400 mg loading
dose of celgosivir followed by 6 subsequent doses of 133 mg of
celgosivir given at 8 hour intervals. 8E shows a simulated
population mean castanospermine exposure after a 300 mg loading
dose of celgosivir followed by 3 subsequent doses of 300 mg of
celgosivir given at 12 hour intervals. 8F shows a simulated
population mean castanospermine exposure after a 200 mg loading
dose of celgosivir followed by 5 subsequent doses of 200 mg of
celgosivir given at 8 hour intervals. 8G shows a simulated
population mean castanospermine exposure after a 150 mg loading
dose of celgosivir followed by 7 subsequent doses of 150 mg of
celgosivir given at 6 hour intervals. 8H shows a simulated
population mean castanospermine exposure after a single dose of 600
mg. For FIGS. 8A-8H the solid line represents the simulated dosing
regimen, the dotted line represents a dosing regimen of 400 mg
initial dose followed by 200 mg every 12 hours for 9 doses (i.e. 5
days of treatment) and the dashed line represents target minimum
trough concentration. LD is loading dose. q is `every` and refers
to the duration of time between doses.
DETAILED DESCRIPTION OF THE INVENTION
A description of example embodiments of the invention follows.
Definitions
All definitions of substituents set forth below are further
applicable to the use of the term in conjunction with another
substituent. Unless defined otherwise, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this invention
belongs.
"Alkyl" as used alone or as part of a larger moiety as in
"arylalkyl" or "aryloxyalkyl" means a saturated aliphatic branched
or straight-chain monovalent hydrocarbon radicals, typically
C1-C16, preferably C1-C12. For example, "(C1-C6) alkyl" means a
radical having from 1-6 carbon atoms in a linear or branched
arrangement. "(C1-C6) alkyl" includes methyl, ethyl, propyl, butyl,
tert-butyl, pentyl and hexyl.
"Alkylene" means a saturated aliphatic straight-chain divalent
hydrocarbon radical. Thus, "(C.sub.1-C.sub.6) alkylene" means a
divalent saturated aliphatic radical having from 1-6 carbon atoms
in a linear arrangement. "(C.sub.1-C.sub.6) alkylene" includes
methylene, ethylene, propylene, butylene, pentylene and
hexylene.
"Cycloalkyl" means saturated aliphatic cyclic hydrocarbon ring.
Thus, "C.sub.3-C.sub.8 cycloalkyl" means (3-8 membered) saturated
aliphatic cyclic hydrocarbon ring. C.sub.3-C.sub.8 cycloalkyl
includes, but is not limited to cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Preferably,
cycloalkyl is C.sub.3-C.sub.6 cycloalkyl.
The term "alkoxy" means --O-alkyl; "arylalkoxy" means an alkoxy
group substituted at any carbon by an aryl group; "hydroxyalkyl"
means alkyl substituted with hydroxy; "arylalkyl" means alkyl
substituted with an aryl group; "alkoxyalkyl" mean alkyl
substituted with an alkoxy group; "cycloalkylalkyl" means alkyl
substituted with cycloalkyl; "alkylcarbonyl" means --C(O)-A*,
wherein A* is alkyl; "alkoxycarbonyl" means --C(O)--OA*, wherein A*
is alkyl; and where alkyl is as defined above. Alkoxy is preferably
O(C.sub.1-C.sub.6) alkyl and includes methoxy, ethoxy, propoxy,
butoxy, pentoxy and hexoxy.
"Cycloalkoxy" means a cycloalkyl-O-- group wherein the cycloalkyl
is as defined above. Exemplary (C.sub.3-C.sub.7) cycloalkyloxy
groups include cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexoxy
and cycloheptoxy.
"Halogen" and "halo" are interchangeably used herein and each
refers to fluorine, chlorine, bromine, or iodine.
The terms "haloalkyl", "halocycloalkyl" and "haloalkoxy" mean
alkyl, cycloalkyl, or alkoxy, as the case may be, substituted with
one or more halogen atoms. The term "halogen" or "halo" means F,
Cl, Br or I.
"Acyl" refers to R''--C(O)--, where R'' is H, alkyl, substituted
alkyl, heteroalkyl, substituted heteroalkyl, alkenyl, substituted
alkenyl, aryl, alkylaryl, or substituted alkylaryl, and is
indicated in the general formula of a particular embodiment as
"Ac".
An "alkylene group" is represented by --[CH.sub.2].sub.z--, wherein
z is a positive integer, preferably from one to eight, more
preferably from one to four.
An "alkenylene group" is an alkylene in which at least a pair of
adjacent methylenes are replaced with --CH.dbd.CH--.
The term "(C.sub.6-C.sub.10) aryl" used alone or as part of a
larger moiety as in "arylalkyl", "arylalkoxy", "aryloxy", or
"aryloxyalkyl", means carbocyclic aromatic rings. The term
"carbocyclic aromatic group" may be used interchangeably with the
terms "aryl", "aryl ring" "carbocyclic aromatic ring", "aryl group"
and "carbocyclic aromatic group". An aryl group typically has 6-10
ring atoms. A "substituted aryl group" is substituted at any one or
more substitutable ring atom. The term "C.sub.6-C.sub.16 aryl" as
used herein means a monocyclic, bicyclic or tricyclic carbocyclic
ring system containing from 6 to 16 carbon atoms and includes
phenyl (Ph), naphthyl, anthracenyl, 1,2-dihydronaphthyl,
1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the
like. The (C.sub.6-C.sub.10) aryl (C.sub.1-C.sub.6) alkyl group
connects to the rest of the molecule through the (C.sub.1-C.sub.6)
alkyl portion of the (C.sub.6-C.sub.10) aryl (C.sub.1-C.sub.6)
alkyl group.
The term "Alkenyl" as used alone or as part of a larger moiety as
in "Alkenylacyl" or "haloalkylacyl" means a straight or branched
hydrocarbon radical having a specified number of carbon atoms and
includes at least one double bond. An alkenyl group generally has
between 2 and 6 carbon atoms. The (C.sub.6-C.sub.10) aryl
(C.sub.2-C.sub.6) alkenyl group connects to the remainder of the
molecule through the (C.sub.2-C.sub.6) alkenyl portion of
(C.sub.6-C.sub.10) aryl (C.sub.2-C.sub.6) alkenyl.
"Alkenylacyl" refers to an acyl group, R''--C(O)--, where R'' is an
alkenyl or a substituted alkenyl (e.g.,
CH.sub.3--CH.dbd.CH--C(O)--).
"Pharmaceutically acceptable carrier" means non-therapeutic
components that are of sufficient purity and quality for use in the
formulation of a composition of the invention that, when
appropriately administered to typically do not produce an adverse
reaction, and that are used as a vehicle for a drug substance
(e.g., a compound of Formula (I)).
"Intraperitoneal injection," as used herein, refers to the
injection of a substance into the peritoneum (body cavity).
"Three times a day dosing", as used herein, refers to three
administrations of a pharmaceutical composition per every 24 hour
period.
"Four times a day dosing" (QDS), as used herein, refers to four
administrations of a pharmaceutical composition per every 24 hour
period.
As used herein, "BDI" refers to twice daily. Further, as used
herein, "QD" refers to once daily.
As used herein, "Cmin" refers to the minimum concentration that a
drug achieves after the drug has been administered and prior to the
administration of a second or additional dose. Further, "Cmax", as
used herein, refers to the maximum concentration. Similarly,
"Tmax", as used herein, refers to the time of maximum
concentration. Additionally, "AUC", used herein, is the area under
the concentration-time curve. Additionally, "50% effective
concentration" (EC50), as used herein, refers to the concentration
of an anti-viral that produces 50% of the maximal possible
antiviral effect.
As used herein, the term "about" refers to a number that differs
from the given number by less than 10%. In other embodiments, the
term "about" indicates that the number differs from the given
number by less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.
The abbreviation "DENV", as used herein, refers to dengue virus.
Further the four scrotypes of the dengue virus are used herein as
"DENV1", "DENV2", "DENV3" and "DENV4".
"Antibody enhanced" or "Antibody Dependent Enhancement" (ADE), as
used herein interchangeably, refers to a DENV infection made more
severe due to a prior infection with one of the four DENV
serotypes: DENV1, DENV2, DENV3, and DENV4.
As used herein, "dengue hemorrhagic fever" (DHF) and "dengue shock
syndrome" (DSS), refers to the severe and potentially fatal dengue
diseases that often occur during secondary heterotypic
infections.
As used herein, "viral load" refers to the amount of virus in the
blood stream of a human subject.
Dosing Regimen
The present invention pertains to methods of treating a dengue
virus (DENV) infection in a human subject, comprising administering
to the human subject an initial dose of about 100 to about 600 mg
of a compound of Formula (I), or a pharmaceutical composition
comprising a compound of Formula (I), within from about onset of
fever to about 72 hours of fever onset due to dengue infection and
administering to the human subject a dose of about 25 to about 300
mg of a compound of Formula (I), or a pharmaceutical composition
comprising a compound of Formula (I), at intervals of from about 6
to about 12 hours until there is an improvement in the infection or
between from about 1 day to about 10 days.
In one embodiment, the compound of the invention is a compound of
Formula (I):
##STR00003##
or a pharmaceutically acceptable salt there of;
wherein R.sup.1, R.sup.2, and R.sup.3 are independently H, (C1-C14)
acyl, (C1-C14) alkenylacyl, (C3-C8) cycloalkylacyl, (C1-C14)
haloalkylacyl (C1-C8) alkoxyacyl, or (C6-C10) arylacyl.
In another embodiment, R.sup.1 and R.sup.2 are H and R.sup.3 is a
(C1-C14) acyl. In another embodiment, R.sup.1 is
CH.sub.3--CH.sub.2CH.sub.2--C(O)--. In yet another embodiment,
R.sup.2 is CH.sub.3--CH.sub.2CH.sub.2--C(O)--. In a further
embodiment, R.sup.3 is CH.sub.3--CH.sub.2CH.sub.2--C(O)--. In
another embodiment, at least one but not more than two R1, R2, and
R3 is a hydrogen.
In yet another embodiment, the compound of the invention is a
compound of Formula (II):
##STR00004## or a pharmaceutically acceptable salt thereof.
The compound of Formula (II), or pharmaceutical composition
comprising a compound of Formula (II), can be used in any of the
embodiments provided herein for Formula (I).
In farther embodiment, the compound of the invention is a compound
of Formula (III):
##STR00005## or a pharmaceutically acceptable salt thereof;
wherein R.sup.1, R.sup.2, and R.sup.3 are independently H, (C1-C14)
acyl, (C1-C14) alkenylacyl, (C3-C8) cycloalkylacyl, (C1-C14)
haloalkylacyl (C1-C8) alkoxyacyl, or (C6-C10) arylacyl.
The compound of Formula (III), or pharmaceutical composition
comprising a compound of Formula (III), can be used in any of the
embodiments provided herein for Formula (I).
The compounds of the invention useful for practicing the methods
described herein may possess one or more chiral centers and so
exist in a number of stereoisomeric forms. All stereoisomers and
mixtures thereof are included in the scope of the present
invention. Racemic compounds may either be separated using
preparative HPLC and a column with a chiral stationary phase or
resolved to yield individual enantiomers methods known to those
skilled in the art. In addition, chiral intermediate compounds may
be resolved and used to prepare chiral compounds of the
invention.
The compounds described herein may exist in one or more tautomeric
forms. All tautomers and mixtures thereof are included in the scope
of the present invention.
The compounds of the present invention can be administered as the
free base or as a pharmaceutically acceptable salt. For example, an
acid salt of a compound of the present invention containing an
amine or other basic group can be obtained by reacting the compound
with a suitable organic or inorganic acid, resulting in
pharmaceutically acceptable anionic salt forms. Examples of anionic
salts include the acetate, benzenesulfonate, benzoate, bicarbonate,
bitartrate, bromide, calcium edetate, camsylate, carbonate,
chloride, citrate, dihydrochloride, edetate, edisylate, estotate,
esylate, fumarate, glyceptate, gluconate, glutamate,
glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride,
hydroxynaphthoate, iodide, isethionate, lactate, lactobionate,
malate, maleate, mandelate, mesylate, methylsulfate, mucate,
napsylate, nitrate, pamoate, pantothenate, phosphate/diphosphate,
polygalacturonate, salicylate, stearate, subacetate, succinate,
sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide
salts. In one embodiment, the compound of Formula (I) is a
hydrochloride salt. In another embodiment, the compound of Formula
(II) is a hydrochloride salt.
The invention is also directed to methods of the invention using a
pharmaceutical composition comprising a compound of Formula (I) or
Formula (II). The disclosed compounds of Formula (I) and Formula
(II) can be administered to the subject in conjunction with an
acceptable pharmaceutical carrier or diluent as part of a
pharmaceutical composition for treatment of a dengue (DENV)
infection, and according to any of the dosing regimens described
herein. Formulation of the compound to be administered will vary
according to the route of administration selected (e.g., solution,
emulsion, capsule). Suitable pharmaceutical carriers may contain
inert ingredients which do not interact with the compound. Standard
pharmaceutical formulation techniques can be employed, such as
those described in Remington's Pharmaceutical Sciences, Mack
Publishing Company, Easton, Pa. Suitable pharmaceutical carriers
for parenteral administration include, for example, sterile water,
physiological saline, bacteriostatic saline (saline containing
about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's
solution, Ringer's-lactate and the like. Methods for encapsulating
compositions (such as in a coating of hard gelatin or cyclodextran)
are known in the art (Baker, et al., "Controlled Release of
Biological Active Agents", John Wiley and Sons, 1986).
In one embodiment, the pharmaceutical composition comprises a
pharmaceutically acceptable carrier or diluent and a compound
Formula (I). In another embodiment, the pharmaceutical composition
comprises a pharmaceutically acceptable carrier or diluent and a
compound Formula (II).
In preferred embodiments, the compound of Formula (I) is prodrug of
castanospermine, a natural product derived from the seeds of
Castanospermum austral. Once administered compounds of Formula (I)
are rapidly converted to castanospermine. Compounds of Formula (I)
(e.g., celgosivir) are more rapidly and efficiently absorbed than
castanospermine. Compounds of Formula (I) are also more readily
absorbed into cells. As a result, compounds of Formula (I) may have
higher EC50 values and in vivo efficacy than castanospermine
against the dengue (DENV) virus.
Castanospermine has been shown to exert antiviral activity by
inhibiting host alpha-glucosidases I and II, enzymes essential for
proper folding of dengue-virus encoded glycoproteins such as E and
prM. (6,7) Castanospermine targets dengue NS1 protein folding in
dengue virus infected cells. Impaired glycosylation of the NS1
protein leads to accumulation of proteins in the endoplasmic
reticulum (ER) and drastically inhibits the virus replication.
Since the drug target is a host enzyme required for viral
maturation, the potential for development of resistance is expected
to be lower than a drug directed against a viral enzyme.
The methods of the invention treat a human subject having a dengue
viral infection. As used herein "treating" or "treatment" refers to
obtaining desired pharmacological and/or physiological effects. The
effect can include achieving, partially or substantially one or
more of the following results: partially or totally reducing the
extent of the disease, disorder or syndrome; ameliorating or
improving a clinical symptom or indicator associated with the
disease, disorder or syndrome; delaying, inhibiting or decreasing
the likelihood of the progression of the disease, disorder or
syndrome.
The initial dose of a compound of Formula (I), or a pharmaceutical
composition comprising a compound of Formula (I), can be
administered at any time within from about the onset of fever to
about 72 hours after fever onset due to dengue infection. In one
embodiment, the initial dose is administered at the time of fever
onset. In another embodiment, the initial dose is administered
within from about 24 hours of fever onset. In yet another
embodiment, the initial dose is administered within from about 48
hours of fever onset. In a further embodiment, the initial dose is
administered within from about 72 hours of fever onset. Subsequent
doses can be the same amount or vary to achieve steady state Cmin
or plasma concentrations in the subject.
The human subject may be an adult or a child. As used herein, a
"child" refers to a human subject who is between the ages of 1 day
to 17 years of age. The term "adult" refers to a human subject who
is 18 years of age or older. Further, the plurality of human
subjects may include adults or children. In some embodiments, the
plurality of human subjects may include only adults. In another
embodiment, the plurality of human subjects may include only
children. In yet another embodiment, the plurality of human
subjects may include both adults and children.
In another aspect, the present invention relates to a method of
treating a dengue invention in an adult subject comprising
administering to the adult subject an initial dose of about 100 to
about 600 mg of a compound of Formula (I), or a pharmaceutical
composition comprising a compound of Formula (I), within from about
onset of fever to about 72 hours of fever onset due to dengue
infection and administering to the adult subject a dose of about 25
to about 300 mg of a compound of Formula (I), or a pharmaceutical
composition comprising a compound of Formula (I), at intervals of
from about 6 to about 12 hours until there is an improvement in the
infection or between about 1 day to about 10 days.
Example embodiments of initial and subsequent doses in an adult are
shown in Table 1:
TABLE-US-00001 TABLE 1 Dosing Regimen for an Adult Initial dose
Subsequent Embodiment (mg).sup.1 dose (mg).sup.2,3 1 100 100 2 150
100 3 200 100 4 250 100 5 300 100 6 350 100 7 400 100 8 450 100 9
500 100 10 550 100 11 600 100 12 100 150 13 150 150 14 200 150 15
250 150 16 300 150 17 350 150 18 400 150 19 450 150 20 500 150 21
550 150 22 600 150 23 600 175 24 100 200 25 150 200 26 200 200 27
250 200 28 300 200 29 350 200 30 400 200 31 450 200 32 500 200 33
550 200 34 600 200 35 100 250 36 150 250 37 200 250 38 250 250 39
300 250 40 350 250 41 400 250 42 450 250 43 500 250 44 500 250 45
550 250 46 600 250 47 100 300 48 150 300 49 200 300 50 250 300 51
300 300 52 350 300 53 400 300 54 450 300 55 450 300 56 500 300 57
500 300 58 550 300 59 600 300 .sup.1Initial dosing within about
onset of fever to about 72 hours. .sup.2Subsequent dosing
periodically from about 6 to about 12 hours. .sup.3Length of
subsequent dosing, a) until infection improves, b) between about 1
day to 2 days, c) between about 2 to 3 days, d) between about 3 to
4 days, d) between about 4 to 5 days, e) between about 5 to 7 day,
or f) between about 7 to 10 days.
In another aspect, the present invention relates to methods of
treating a dengue invention in an child subject comprising
administering to the child subject an initial dose of about 100 to
about 600 mg of a compound of Formula (I), or a pharmaceutics
composition comprising a compound of Formula (I), within from about
onset of fever to about 72 hours of fever onset due to dengue
infection and administering to the child subject a dose of about 25
to about 300 mg of a compound of Formula (I), or a pharmaceutical
composition comprising a compound of Formula (I), at intervals of
from about 6 to about 12 hours until there is an improvement in the
infection or between from about 1 day to about 10 days.
Example embodiments of initial and subsequent doses combinations in
a child are shown in Table 2:
TABLE-US-00002 TABLE 2 Dosing Regimen for a Child Initial dose
Subsequent Embodiment (mg).sup.4 dose (mg).sup.5,6 1 25 25 2 50 25
3 75 25 4 100 25 5 150 25 6 200 25 7 250 25 8 300 25 9 25 50 10 50
50 11 75 50 12 100 50 13 150 50 14 200 50 15 250 50 16 300 50 17 25
75 18 50 75 19 75 75 20 100 75 21 150 75 22 200 75 23 250 75 24 300
75 25 25 100 26 50 100 27 75 100 28 100 100 29 150 100 30 200 100
31 250 100 32 300 100 33 25 150 34 50 150 35 75 150 36 100 150 37
150 150 38 200 150 39 250 150 40 300 150 41 25 200 42 50 200 43 75
200 44 100 200 45 150 200 46 200 200 47 250 200 48 300 200
.sup.4Initial dosing within about onset of fever to about 72 hours.
.sup.5Subsequent dosing periodically from about 6 to about 12
hours. .sup.6Length of subsequent dosing, a) until infection
improves, b) between about 1 day to 2 days, c) between about 2 to 3
days, d) between about 3 to 4 days, d) between about 4 to 5 days,
e) between about 5 to 7 day, or f) between about 7 to 10 days.
The human subject can be administered a compound of the present
invention for a period of about between about 1 day to about 10
days. In one embodiment, the subsequent dose of a compound of
Formula (I), or a pharmaceutical composition comprising a compound
of Formula (I), is administered for about 1 day to about 2 days. In
further embodiment, the subsequent dose of a compound of Formula
(I), or a pharmaceutical composition comprising a compound of
Formula (I), is administered for about 2 to about 3 days. In
another embodiment, the subsequent dose of a compound of Formula
(I), or a pharmaceutical composition comprising a compound of
Formula (I), is administered for about 3 to about 5 days. In yet
another embodiment, the subsequent dose of a compound of Formula
(I), or a pharmaceutical composition comprising a compound of
Formula (I), is administered for about 5 to about 7 days. In
another embodiment, the subsequent dose of a compound of Formula
(I), or a pharmaceutical composition comprising a compound of
Formula (I), is administered for about 7 to about 10 days.
The present invention pertains to methods of treating a dengue
virus (DENV) infection in a human subject, comprising administering
to the human subject an initial dose of about 100 to about 600 mg
of a compound of Formula (I), or a pharmaceutical composition
comprising a compound of Formula (I), within from about onset of
fever to about 72 hours of fever onset due to dengue infection; and
administering to the human subject a dose of about 25 to about 300
mg of a compound of Formula (I), or a pharmaceutical composition
comprising a compound of Formula (I), at intervals of from about 6
to about 12 hours until there is an improvement in the infection or
between from about 1 day to about 10 days, wherein R.sup.1,
R.sup.2, and R.sup.3 are independently H, (C1-C14) acyl, (C1-C14)
alkenylacyl, (C3-C8) cycloalkylacyl, (C1-C14) haloalkylacyl (C1-C8)
alkoxyacyl, or (C6-C10) arylacyl.
The invention is also directed to methods of treating a secondary
dengue infection in a human subject, comprising administering to
the human subject an initial dose of about 100 to about 600 mg of a
compound of Formula (I), or a pharmaceutical composition comprising
a compound of Formula (I), within from about onset of fever to
about 72 hours of fever onset due to dengue infection; and
administering to the human subject a dose of about 25 to about 300
mg of a compound of Formula (I), or a pharmaceutical composition
comprising a compound of Formula (I), at intervals of from about 6
to about 12 hours until there is an improvement in the infection or
between from about 1 day to about 10 days, wherein R.sup.1,
R.sup.2, and R.sup.3 are independently H, (C1-C14) acyl, (C1-C14)
alkenylacyl, (C3-C8) cycloalkylacyl, (C1-C14) haloalkylacyl (C1-C8)
alkoxyacyl, or (C6-C10) arylacyl.
In one embodiment of the invention, the compound of Formula (I), is
a compound of Formula (II) or a pharmaceutically acceptable salt
thereof.
The invention also relates to methods of treating a dengue virus
(DENV) infection in a human subject, comprising administering to
the human subject an initial dose of about 100 to about 600 mg of a
compound of Formula (II), or a pharmaceutical composition
comprising a compound of Formula (II), within from about onset of
fever to about 72 hours of fever onset due to dengue infection; and
administering to the human subject a dose of about 25 to about 300
mg of a compound of Formula (II), or a pharmaceutical composition
comprising a compound or Formula (II), at intervals of from about 6
to about 12 hours until there is an improvement in the infection or
between from about 1 day to about 10 days.
In another aspect, the invention relates to methods of treating a
dengue virus (DENV) infection in a human subject, comprising
administering orally to the human subject an initial dose of about
100 to about 600 mg of a compound of Formula (I), or a
pharmaceutical composition comprising a compound of Formula (I),
within from about onset of fever to about 72 hours of fever onset
due to dengue infection; and administering orally to the human
subject a dose of about 25 to about 300 mg of a compound of Formula
(I), or a pharmaceutical composition comprising a compound of
Formula (I), at intervals of from about 6 to about 12 hours until
there is an improvement in the infection or between from about 1
day to about 10 days, wherein R.sup.1, R.sup.2, and R.sup.3 are
independently H, (C1-C14) acyl, (C1-C14) alkenylacyl, (C3-C8)
cycloalkylacyl, (C1-C14) haloalkylacyl (C1-C8) alkoxyacyl, or
(C6-C10) arylacyl.
In yet another aspect, the invention pertains to methods of
treating a dengue virus (DENV) infection in a human subject,
comprising administering to the human subject an initial dose of
about 100 to about 600 mg of a compound of Formula (III), or a
pharmaceutical composition comprising a compound of Formula (III),
within from about onset of fever to about 72 hours of fever onset
due to dengue infection; and administering to the human subject a
dose of about 25 to about 300 mg of a compound of Formula (III), or
a pharmaceutical composition comprising a compound or Formula
(III), at intervals of from about 6 to about 12 hours until there
is an improvement in the infection or between from about 1 day to
about 10 days; wherein R.sup.1, R.sup.2, and R.sup.3 are
independently H, (C1-C14) acyl, (C1-C14) alkenylacyl, (C3-C8)
cycloalkylacyl, (C1-C14) haloalkylacyl (C1-C8) alkoxyacyl, or
(C6-C10) arylacyl.
In certain embodiments of the invention, the human subject is an
adult or a child. In further embodiments of the invention, the
plurality of human subjects may include adults or children. In some
embodiments, the plurality of human subjects may include only
adults. In another embodiment, the plurality of human subjects may
include only children. In yet another embodiment, the plurality of
human subjects may include both adults and children.
In certain embodiments of the invention, each of the plurality of
human subjects may be given a different dose. In another
embodiment, each of the plurality of human subjects may be given
the same dose. In further embodiment, each of the plurality of
human subjects may be given a variety of doses. In yet another
embodiment, some of the plurality of the human subjects may be
given the same dose and some of the plurality of the human subjects
may be given a different dose.
In another embodiment, compound of Formula (I), the compound of
Formula (II), or the compound of Formula (III) is converted to
castanospermine after administration to a human subject.
In yet another embodiment, a steady state Cmin serum or plasma
concentration of between about 0.05 and about 2.0 microgram/mL of
castanospermine in an adult or child human subject is attained.
In one embodiment of the invention, dengue viral infection
comprises at least one dengue virus selected from DENV1, DENV2,
DENV3 and DENV4. In another embodiment, the dengue viral infection
is secondary dengue infection. In yet another embodiment of the
invention, the human subject to be treated is positive for a dengue
infection using a NS1 strip assay or quantitative PCR. In further
embodiment of the invention, the virological log reduction in
treated human subjects is at least 50% greater than untreated or
placebo-treated groups. In yet another embodiment, administering
the compound, or the pharmaceutical composition, achieves a steady
state Cmin serum or plasma concentration of between about 0.05 and
about 2.0 microgram/mL of castanospermine. In another embodiment of
the invention, the compound, or the pharmaceutical composition, is
administered intravenously, orally, rectally or sublingually.
In one embodiment of the invention, the human subject is
administered an initial dose of 150 mg and a dose of 100 mg is
administered to the human subject every 6 hours for about 1 day. In
another embodiment, the human subject is administered an initial
dose, of 150 mg and a dose of 100 mg is administered to the human
subject every 6 hours for about 2 days. In yet another embodiment,
the human subject is administered an initial dose of 150 mg and a
dose of 100 mg is administered to the human subject every 6 hours
for about 5 days. In further embodiment, the human subject is
administered an initial dose of 150 mg and a dose of 100 mg is
administered to the human subject every 8 hours for about 1 day. In
certain embodiment, the human subject is administered an initial
dose of 150 mg and a dose of 100 mg is administered to the human
subject every 8 hours for about 2 days. In another embodiment, the
human subject is administered an initial dose of 150 mg and a dose
of 100 mg is administered to the human subject every 8 hours for
about 5 days. In yet another embodiment, the human subject is
administered an initial dose of 150 mg and a dose of 100 mg is
administered to the human subject every 12 hours for about 1 day.
In another embodiment, the human subject is administered an initial
dose of 150 mg and a dose of 100 mg is administered to the human
subject every 12 hours for about 2 days. In further embodiment, the
human subject is administered an initial dose of 150 mg and a dose
of 100 mg is administered to the human subject every 12 hours for
about 5 days. In yet another embodiment, the human subject is
administered an initial dose of 150 mg and a dose of 150 mg is
administered to the human subject every 6 hours for about 1 day. In
another embodiment, the human subject is administered an initial
dose of 150 mg and a dose of 150 mg is administered to the human
subject every 6 hours for about 2 days. In yet another embodiment,
the human subject is administered an initial dose of 150 mg and a
dose of 150 mg is administered to the human subject every 6 hours
for about 5 days. In another embodiment, the human subject is
administered an initial dose of 150 mg and a dose of 150 mg is
administered to the human subject every 8 hours for about 1 day. In
certain embodiment, the human subject is administered an initial
dose of 150 mg and a dose of 150 mg is administered to the human
subject every 8 hours for about 2 days. In yet another embodiment,
the human subject is administered an initial dose of 150 mg and a
dose of 150 mg is administered to the human subject every 8 hours
for about 5 days. In one embodiment, the human subject is
administered an initial dose of 150 mg and a dose of 150 mg is
administered to the human subject every 12 hours for about 1 day.
In further embodiment, the human subject is administered an initial
dose of 150 mg and a dose of 150 mg is administered to the human
subject every 12 hours for about 2 days. In yet another embodiment,
the human subject is administered an initial dose of 150 mg and a
dose of 150 mg is administered to the human subject every 12 hours
for about 5 days. In another embodiment, the human subject is
administered an initial dose of 150 mg and a dose of 200 mg is
administered to the human subject every 6 hours for about 1 day. In
one embodiment, the human subject is administered an initial dose
of 150 mg and a dose of 200 mg is administered to the human subject
every 6 hours for about 2 days. In another embodiment, the human
subject is administered an initial dose of 150 mg and a dose of 200
mg is administered to the human subject every 6 hours for about 5
days. In further embodiment, wherein the human subject is
administered an initial dose of 150 mg and a dose of 200 mg is
administered to the human subject every 8 hours for about 1 day. In
yet another embodiment, the human subject is administered an
initial dose of 150 mg and a dose of 200 mg is administered to the
human subject every 8 hours for about 2 days. In one embodiment,
the human subject is administered an initial dose of 150 mg and a
dose of 200 mg is administered to the human subject every 8 hours
for about 5 days. In another embodiment, the human subject is
administered an initial dose of 150 mg and a dose of 200 mg is
administered to the human subject every 12 hours for about 1 day.
In further embodiment, the human subject is administered an initial
dose of 150 mg and a dose of 200 mg is administered to the human
subject every 12 hours for about 2 days. In another embodiment, the
human subject is administered an initial dose of 150 mg and a dose
of 200 mg is administered to the human subject every 12 hours for
about 5 days. In certain embodiment, the human subject is
administered an initial dose of 200 mg and a dose of 100 mg is
administered to the human subject every 6 hours for about 1 day. In
yet another embodiment, the human subject is administered an
initial dose of 200 mg and a dose of 100 mg is administered to the
human subject every 6 hours for about 2 days. In another
embodiment, the human subject is administered an initial dose of
200 mg and a dose of 100 mg is administered to the human subject
every 6 hours for about 5 days. In one embodiment, the human
subject is administered an initial dose of 200 mg and a dose of 100
mg is administered to the human subject every 8 hours for about 1
day. In further embodiment, the human subject is administered an
initial dose of 200 mg and a dose of 100 mg is administered to the
human subject every 8 hours for about 2 days. In yet another
embodiment, the human subject is administered an initial dose of
200 mg and a dose of 100 mg is administered to the human subject
every 8 hours for about 5 days. In another embodiment, the human
subject is administered an initial dose of 200 mg and a dose of 100
mg is administered to the human subject every 12 hours for about 1
day. In one embodiment, the human subject is administered an
initial dose of 200 mg and a dose of 100 mg is administered to the
human subject every 12 hours for about 2 days. In another
embodiment, the human subject is administered an initial dose of
200 mg and a dose of 100 mg is administered to the human subject
every 12 hours for about 5 days. In further embodiment, the human
subject is administered an initial dose of 200 mg and a dose of 150
mg is administered to the human subject every 6 hours for about 1
day. In another embodiment of the invention, the human subject is
administered an initial dose of 200 mg and a dose of 150 mg is
administered to the human subject every 6 hours for about 2 days.
In yet another embodiment, the human subject is administered an
initial dose of 200 mg and a dose of 150 mg is administered to the
human subject every 6 hours for about 5 days. In another
embodiment, the human subject is administered an initial dose of
200 mg and a dose of 150 mg is administered to the human subject
every 8 hours for about 1 day. In one embodiment, the human subject
is administered an initial dose of 200 mg and a dose of 150 mg is
administered to the human subject every 8 hours for about 2 days.
In yet another embodiment, the human subject is administered an
initial dose of 200 mg and a dose of 150 mg is administered to the
human subject every 8 hours for about 5 days. In another
embodiment, the human subject is administered an initial dose of
200 mg and a dose of 150 mg is administered to the human subject
every 12 hours for about 1 day. In another embodiment, the human
subject is administered an initial dose of 200 mg and a dose of 150
mg is administered to the human subject every 12 hours for about 2
days. In an embodiment of the invention, the human subject is
administered an initial dose of 200 mg and a dose of 150 mg is
administered to the human subject every 12 hours for about 5 days.
In further embodiment, the human subject is administered an initial
dose of 200 mg and a dose of 200 mg is administered to the human
subject every 6 hours for about 1 day. In yet another embodiment,
the human subject is administered an initial dose of 200 mg and a
dose of 200 mg is administered to the human subject every 6 hours
for about 2 days. In another embodiment, the human subject is
administered an initial dose of 200 mg and a dose of 200 mg is
administered to the human subject every 6 hours for about 5 days.
In an embodiment of the invention, the human subject is
administered an initial dose of 200 mg and a dose of 200 mg is
administered to the human subject every 8 hours for about 1 day. In
further embodiment, the human subject is administered an initial
dose of 200 mg and a dose of 200 mg is administered to the human
subject every 8 hours for about 2 days. In yet another embodiment,
the human subject is administered an initial dose of 200 mg and a
dose of 200 mg is administered to the human subject every 8 hours
for about 5 days. In another embodiment, the human subject is
administered an initial dose of 200 mg and a dose of 200 mg is
administered to the human subject every 12 hours for about 1 day.
In an embodiment of the invention, the human subject is
administered an initial dose of 200 mg and a dose of 200 mg is
administered to the human subject every 12 hours for about 2 days.
In one embodiment, the human subject is administered an initial
dose of 200 mg and a dose of 200 mg is administered to the human
subject every 12 hours for about 5 days. In another embodiment, the
human subject is administered a single or a divided dose of about
25 to about 300 mg of the compound or the pharmaceutical
composition, for about between about 5 days to about 10 days. In
yet another embodiment, the human subject is administered a divided
dose of about 25 to about 300 mg of the compound, or the
pharmaceutical composition, for about between about 5 days to about
10 days.
The compounds of the present invention can be administered in a
single or a divided dose. In one embodiment, the human subject is
administered a divided dose of about 25 to about 300 mg of a
compound of Formula (I), or a pharmaceutical composition comprising
a compound of Formula (I), for about between about 1 day to about
10 days. In another embodiment, the human subject is administered a
single dose of about 25 to about 300 mg of a compound of Formula
(I), or a pharmaceutical composition comprising a compound of
Formula (I), for about between about 1 day to about 10 days.
Compounds of Formula (I) and Formula (II) inhibit the replication
of a variety of laboratory and clinical dengue strains of DENV1-4,
with submicromolar EC50 values. As used herein, "EC50" refers to
the concentration of an anti-viral that produces 50% of the maximal
possible antiviral effect. In one embodiment, the method of the
invention is used to treat an infection caused by at least one
dengue virus selected from DENV1, DENV2, DENV3, and DENV4.
The method of the present invention is used to treat a dengue viral
infection in a subject who has tested positive for a dengue virus.
Known methods for diagnosis of dengue viral infection can be used
including, but not limited to, an NS1 strip assay or a quantitative
PCR assay. The selected method should be rapid enough for a
diagnosis within from about onset of fever to about 72 hours of
fever onset to optimize the therapeutic regimen of the various
embodiments of the invention.
In one embodiment, the human subject tests positive for a dengue
infection in a NS1 strip assay. In another embodiment, the human
subject tests positive for a dengue infection in a quantitative PCR
assay.
The compounds of the present invention can be administered
intravenously, orally, rectally or sublingually. Intravenous, oral,
rectal and sublingual dosing can be in a single or divided dose.
Intravenous dosing can also be a slow infusion over a period of
time and the slow infusions can be constant or intermittent.
The compounds of the invention can be administered several times a
day or as needed to maintain a steady Cmin serum concentration of
between about 0.05 and about 2.0 microgram/mL. A suitable interval
between the two administrations includes any time period which
maintains a therapeutically effective plasma level of a compound of
Formula (I). Such an interval can be, for example, about 12 hours.
In one embodiment, the human subject is administered a compound of
Formula (I) or a compound of Formula (II) twice a day. Dosing at
intervals is intended to cover subsequent dosing, either as a
single or a divided dose, routinely during the course of therapy.
For example, dosing can be about every 6 to about 12 hours during
the course of treatment, but it is intended to cover the
possibility that a dose may have been missed during at least one
interval. In another embodiment, the human subject is administered
a compound of Formula (I) or a compound of Formula (II) three times
a day. In yet another embodiment, the human subject is administered
a compound of Formula (I) or a compound of Formula (II) four times
a clay.
As such, a first dose can be administered at 6 am on Day 1 and a
second dose can be administered at 6 pm on Day 1 for a total of two
doses in a 24 hour period or day. Further, a first dose can be
administered at 12 am on Day 1, a second dose can be administered
at 6 am on Day 1, a third dose can be administered at 12 pm on Day
1, and a fourth dose can be administered at 6 pm on Day 1 for a
total of four doses in a 24 hour period or day. In one embodiment,
the therapeutically effective plasma level is the level at which
the Cmin concentration of a compound of Formula (I) is
achieved.
The invention also relates to a method of treating a dengue virus
infection by achieving a steady state Cmin serum or plasma
concentration of between about 0.05 and about 2.0 microgram/mL of
castanospermine in an adult or child human subject. As used herein,
"Cmin" refers to the minimum concentration that a drug achieves
after the drug has been administered and prior to the
administration of a second or additional dose. Steady state Cmin is
achieved when the overall intake of a drug Cmin concentration is
fairly in dynamic equilibrium with its elimination. In some
embodiments, Cmin concentration of castanospermine is determined at
one or more points following treatment with techniques known in the
art.
In one embodiment, the steady state Cmin serum or plasma
concentration achieved in an adult or child subject is between
about 0.08 and about 0.5 microgram/mL of castanospermine. In
another embodiment, the steady state Cmin serum or plasma
concentration achieved in an adult or child subject is between
about 0.05 and about 0.08 microgram/mL of castanospermine. In yet
another embodiment, the steady state Cmin serum or plasma
concentration achieved in an adult or child subject is between
about 0.08 and about 0.11 microgram/mL of castanospermine. In a
further embodiment, the steady state Cmin serum or plasma
concentration achieved in an adult or child subject is between
about 0.11 and about 0.3 microgram/mL of castanospermine. In
another embodiment, the steady state Cmin serum or plasma
concentration achieved in an adult or child subject is between
about 0.3 and about 0.75 microgram/mL of castanospermine. In
further embodiment, the steady state Cmin serum or plasma
concentration achieved in an adult or child subject is between
about 0.75 and about 1.0 microgram/mL of castanospermine. In yet
another embodiment, the steady state Cmin serum or plasma
concentration achieved in an adult or child subject is between
about 1.0 and about 2.0 microgram/mL of castanospermine. In another
embodiment, the steady state Cmin serum or plasma concentration
achieved in an adult or child subject is between about 1.0 and
about 1.5 microgram/mL of castanospermine. In further embodiment,
the steady state Cmin serum or plasma concentration achieved in an
adult or child subject is between about 1.5 and about 2.0
microgram/mL of castanospermine. In yet another embodiment, the
steady state Cmin serum or plasma concentration achieved in an
adult or child subject is between about 1.25 and about 1.75
microgram/mL of castanospermine.
The invention further relates to a method of treating a dengue
virus infection by achieving an average steady state Cmin serum or
plasma concentration of between about 0.05 and about 2.0
microgram/mL of castanospermine in a plurality of human subjects
after the administration of the compounds of the invention. The
average steady state Cmin of the plurality of human subjects is
calculated as an average of steady state Cmin serum or plasma
concentrations from each of the plurality of human subjects. In
certain embodiments of the invention, an average steady state Cmin
serum or plasma concentration of between about 0.05 and about 2.0
microgram/mL of castanospermine in the plurality of human subjects
is attained after treatment.
In one embodiment, the average steady state Cmin serum or plasma
concentration achieved in the plurality of human subjects is
between about 0.08 and about 0.5 microgram/mL of castanospermine.
In another embodiment, the average steady state Cmin serum or
plasma concentration achieved in the plurality of human subjects is
between about 0.05 and about 0.08 microgram/mL of castanospermine.
In yet another embodiment, the average steady state Cmin serum or
plasma concentration achieved in the plurality of human subjects is
between about 0.08 and about 0.11 microgram/mL of castanospermine.
In a further embodiment, the average steady state Cmin serum or
plasma concentration achieved in the plurality of human subjects is
between about 0.11 and about 0.3 microgram/mL of castanospermine.
In another embodiment, the average steady state Cmin serum or
plasma concentration achieved in the plurality of human subjects is
between about 0.3 and about 0.75 microgram/mL of castanospermine.
In further embodiment, the average steady state Cmin serum or
plasma concentration achieved in the plurality of human subjects is
between about 0.75 and about 1.0 microgram/mL of castanospermine.
In yet another embodiment, the average steady state Cmin serum or
plasma concentration achieved in the plurality of human subjects is
between about 1.0 and about 2.0 microgram/mL of castanospermine. In
another embodiment, the average steady state Cmin serum or plasma
concentration achieved in the plurality of human subjects is
between about 1.0 and about 1.5 microgram/mL of castanospermine. In
further embodiment, the average steady state Cmin serum or plasma
concentration achieved in the plurality of human subjects is
between about 1.5 and about 2.0 microgram/mL of castanospermine. In
yet another embodiment, the average steady state Cmin serum or
plasma concentration achieved in the plurality of human subjects is
between about 1.25 and about 1.75 microgram/mL of
castanospermine.
In another aspect, methods of the present invention can treat a
secondary dengue infection or an "antibody enhanced" (ADE) dengue
infection. A "secondary" infection refers to a DENV infection in a
patient who was previously infected with DENV. An "antibody
enhanced" infection refers to a DENV infection made more severe due
to a prior infection with one of the four DENV serotypes. Ninety
percent (90%) of severe and potentially fatal dengue diseases, such
as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS)
are caused by a secondary dengue infection. In secondary dengue
infection, prior infection causes generation of an antibody that
takes on a pathogenic role. Upon reinfection with DENV, the
antibody response triggers a systemic inflammatory reaction
resulting in vascular leakage. In one embodiment, the dengue viral
infection is a secondary dengue infection.
In another aspect of the invention, viral load reduction of a
treated human subject is at least 50% greater than untreated or
placebo-treated human subjects. As used herein, the term "viral
load" refers to the amount of virus in the blood stream of a human
subject. The viral load is measured before administration of the
first dose and then at various time intervals after administration.
The dose amounts can be adjusted to increase the viral load
reduction or if no viral reduction is observed at a specific dose
it can be adjusted to promote viral reduction.
In one embodiment, the virological log reduction in human subjects
treated with a compound of Formula (I) is at least about 50%
greater than untreated or placebo-treated groups. In another
embodiment, the virological tog reduction in human subjects treated
with a compound of Formula (I) is between about 60 to 70% greater
than untreated or placebo-treated groups. In another embodiment,
the virological log reduction in human subjects treated with a
compound of Formula (I) is between about 70 to 80% greater than
untreated or placebo-treated groups. In yet another embodiment, the
virological log reduction in human subjects treated with a compound
of Formula (I) is between about 80 to 90% greater than untreated or
placebo-treated groups.
EXAMPLES
Example 1
Cell-based Flaviviral Immunodetection Assay (CFI)
BHK21 cells were seeded at 1.3.times.104 in 96-well plate and
incubated overnight at 37.degree. C. in 5% CO.sub.2 incubator.
Confluent monolayer of BHK21 cells were infected with DENV2 (TSV01
strain) at an MOI (multiplicity of infection) 0.3 in the presence
of various concentrations of test compounds and incubated for 1
hour at 37.degree. C. in 5% CO.sub.2 incubator. Infected cells were
incubated with test compounds for another 48 hours at 37.degree. C.
in 5A CO.sub.2. Cells were fixed with methanol and mouse monoclonal
antibody 4G2 was used to detect DENV E protein, which was
quantified using a secondary anti-mouse antibody conjugated with
horseradish peroxidase (HRP). Absorbance was read at 450 nm and
dose-response curve was plotted accordingly. The 50% effective
concentration (EC50), that is, the concentration of the test
compound that decreased the level of viral E protein production by
50%, was calculated by nonlinear regression analysis.
Celgosivir inhibits virus production in a concentration-dependent
manner with an EC50 0.22 .mu.M for DENV2 (FIG. 1). The EC50 values
for the other three serotypes are also in the sub-micromolar range
(0.31 to 0.65 .mu.M). Castanospermine (CAST) has an EC50 of
.about.21 .mu.M against DENV2, which may be attributed to its lower
uptake into cells compared with celgosivir.
The results are shown in Table 3.
TABLE-US-00003 TABLE 3 EC50 values of celgosivir hydrochloride on
DENV 1-4 infection in the CFI assay. Dengue Serotype CFI (EC50)
Compound DENV1 0.65 .+-. 0.16 .mu.M Celgosivir DENV2 0.22 .+-. 0.01
.mu.M Celgosivir DENV3 0.68 .+-. 0.02 .mu.M Celgosivir DENV4 0.31
.+-. 0.12 .mu.M Celgosivir DENV2 ~21 .mu.M Castanospermine
Example 2
Fluorescence Microscopy
BHK21 cells were infected with DENV2 and treated with 20 .mu.M
celgosivir or saline. After 24 hr incubation, cells were stained
with nuclear stain diamidino-2-phenylindole (DAPI) or with a MAb
against NS1 in conjunction with a secondary antibody conjugated
with Alexa-488 (green), then examined by fluorescent microscopy for
the presence of NS1, a marker for viral replication. Viral-infected
cells show abundant NS1 in the cytoplasm, whereas those treated
with celgosivir have almost complete suppression of viral
replication (FIG. 2).
Other cellular microscopic studies, which used specific antibodies
that are markers for the endoplasmic reticulum (ER) or Golgi
(BiP/Grp78) and Gigantin, respectively, were conducted and
demonstrated that nonglycosylated NS1 accumulates in the ER and
does not transport through the trans-Golgi network in order for it
to be processed for secretion.
Celgosivir treatment also results in upregulation of pro-survival
host gene products such as EDEM and XBP1, while apoptotic markers
such as CHOP are down-regulated during drug-induced unfolded
protein response (data not shown). The pro-survival products
enhance the clearance of misfolded proteins by directing them to
the proteosome for degradation.
Example 3
Replicon Assay
The DENV subgenomic replicon was derived from the DENV2 strain NGC
and consists of a luciferase reporter and a puromycin resistance
gene for stable transfection. (7) Only the nonstructural proteins
NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 were represented in the
replicon construct. Of the nonstructural proteins, only NS1 is
known to be glycosylated.
The stably transfected A549 replicon cell line was plated into a
96-well plate in the presence of various concentrations of test
compounds and incubated for 48 hour at 37.degree. C. in 5% CO.sub.2
incubator. The luciferase activity of the replicon treated with
various concentrations of the test compound was measured using
EnduRen (Promega) at a 1:1000 dilution followed by incubation for 2
hrs before the luminescence was read using a Tecan plate-reader.
The data was plotted against the log transformation of the
concentration of the compounds to obtain a dose-response curve to
calculate the EC50 value. Celgosivir inhibited replicon production
in a concentration-dependent manner, with an EC50 value of 2.2
.mu.M (FIG. 3). This data shows inhibition of host alphaglucosidase
activity by celgosivir targets both structural and non-structural
dengue proteins and severely affects viral assembly and
replication.
Example 4
ADE Infection of Human Monocytes
The activity of celgosivir in infected human monocytes, one of the
primary circulating cell types infected by dengue virus, was
examined, THP-1 cells were grown in RPMI-1640 maintenance medium
containing 10% fetal calf serum and 1% Penicillin-streptomycin and
cultured in 37.degree. C. incubator supplemented with 5% CO.sub.2.
For ADE infection in THP-1 cells, virus (DENV2, MOI-10) was mixed
with sub-neutralizing concentration of humanized 4G2 monoclonal
antibody (0.05 .mu.g/0.5 ml) and allowed to incubate on ice for 1
hr in the serum free medium, to allow immune complex formations.
The immune complex was then added on to THP-1 (1.times.10.sup.5)
cells per well in a 24 well plate and further incubated for 2 hours
at 37.degree. C. incubator. An excess unbound immune complex was
then removed by spinning cells at 1000 rpm for 5 min and then cells
were supplemented with complete growth maintenance medium. For drug
testing in the ADE condition, cells were mock (untreated) or
celgosivir-treated for 48 hours, and finally media supernatant was
collected for plaque assay analysis.
Addition of MAb against protein E results in a nearly 2 log
increase in viremia (FIG. 4A). After addition of varying
concentrations of celgosivir, viral replication was suppressed with
an EC50 of 0.5 .mu.M (FIG. 4B). Under the same conditions,
Castanospermine was shown to suppress viral replication with an
EC50 of 14 .mu.M.
Example 5
Animal Models
Mouse Viremia Models
To develop an in vivo test system for anti-dengue drugs,
immunocompetent Sv/129 mice deficient in type I and II interferon
receptors (AG129), were injected with an unadapted, clinical strain
of DENV2, resulting in a dose-dependent transient viremia lasting
several days and peaking on day 3 after infection. (8) NS1 protein,
proinflammatory cytokines, and neutralizing IgM and IgG antibodies
were detected and also the mice had splenomegaly. (8) Selected
compounds such as celgosivir significantly reduced viremia in a
dose-dependent manner, even after delayed treatment, leading to a
reduction of splenomegaly and proinflammatory cytokine levels.
Thereby validating dengue this mouse model as a suitable system for
testing anti-dengue drugs. Furthermore, they indicate that
antiviral treatment during the acute phase of dengue fever can
reduce the severity of the disease. (8) In the AG129 mouse model,
celgosivir significantly reduced viremia by 88% and 55%,
respectively, when given at the time of infection or after
treatment was delayed by 24 hours.
To model ADE, mice were injected IP (intraperitoneal) with 15 mg of
mouse monoclonal antibody against the (DENV) E protein (4G2 clone)
one day prior to infection. (9) For treatment, celgosivir was
injected IP once or twice daily for a total of 5 days. Mice were
monitored every day till Day 12 post infection. The data were
plotted as Kaplan-Meier curves using Prism 5.0 software.
In this lethal model of viremia, untreated mice had 0% survival by
Day 5, whereas mice treated with 50 mg/kg celgosivir PO (oral) BID
(twice a day), showed 100% survival even at Day 12 (p=0.0001). Even
when treatment was delayed 24 and 48 hr, celgosivir was still able
to confer protection, with 75% and 50% survival, respectively at
Day 12. Dosing studies at 10, 25, and 50 mg/kg PO BID resulted in
12%, 62% and 100% survival respectively. Surprisingly when mice
were treated once daily at 100 mg/kg PO, no mice survived past day
6 compared to 100% Survival at 50 mg/kg twice daily, indicating
that the drug, when divided into two doses, is more effective than
giving, the same total dose once a day.
Example 6
Dosing and Schedule Effects
To evaluate the effect of dose and dose schedule, four drug
regimens were studied in the ADE viremia model: 10, 25 and 50 mg/kg
BID and 100 mg/kg QD (once daily). For the BID schedule, survival
increased with increasing dose with survival rates of 13%, 63% and
100% for 10, 25, and 50 mg/kg BID. Survival rates were also
sensitive to schedule -50 mg/kg BID dosing achieved complete
protection, but the same total dose of 100 mg/kg given as a single
daily dose failed to protect animals from death, with none
surviving past Day 6. Even 25 mg/kg given BID was more effective
than 100 mg/kg QD. Hence, dividing the dose was more effective than
a larger daily dose.
Delayed Dosing
Even when treatment was delayed (FIG. 5A), celgosivir (50 mg/kg
BID) exerted a protective effect. In this experiment, survival at
Day 12 was 100%, 75%, and 50% when treatment was immediate, delayed
24 hr or delayed 48 hr after DENV2 infection, respectively, in the
ADE model; by contrast, none (0%) of the sham-treated mice survived
past day 5 (FIG. 5B). Viremia, as determined by plaque assay, in
celgosivir-treated mice (50 mg/kg BID) was 8.3% of levels in
sham-treated mice after only 1 day of dosing. By study day 3,
viremia had increased in untreated mice by more than 7-fold.
Celgosivir demonstrated significantly lower viremia in treated
mice--levels were 17%, 26% and 69% of levels in sham-treated mice
when treatment was immediate, delayed 24 hr, or delayed 48 hr,
respectively. The results also demonstrate that lower viremia is
associated with improved outcome.
Example 7
Pharmacokinetics Mouse Studies
Celgosivir is more rapidly and efficiently absorbed than
castanospermine. In the mouse, single-dose celgosivir (25 mg/kg PC)
demonstrated a 5-fold greater uptake into the plasma at 2 and 5 min
post-dosing than a single dose of Castanospermine (16 mg/kg PO). At
5 min after administration of celgosivir, only castanospermine was
detected in the plasma, confirming the rapid conversion of
celgosivir into castanospermine.
The concentration profile of celgosivir and castanospermine in the
mouse after a single IP dose of celgosivir at a dose of 50 mg/kg is
illustrated in FIG. 6A. Serum concentrations were measured using a
validated LC/MS/MS assay performed. Celgosivir was measurable in
serum only up to 1 hr post-dosing. Conversion to castanospermine is
rapid, and the maximal castanospermine concentration (Cmax) of 31.6
.mu.g/mL occurs at the first sampling time of 10 min post-dosing.
AUC was 22.8 .mu.ghr/ml. Apparent clearance (CL/F) was 2.2 L/h/kg,
mean residence time was 3.5 hr, and the terminal half-life was 5.0
hr.
The castanospermine concentration profile after dosing at 50 mg/kg
BID for 5 days was calculated by nonparametric superposition (FIG.
6B). This dosing regimen of celgosivir protected 100% of mice in
the lethal ADE dengue infection model. Steady state Cmin and Cmax
were 0.4 .mu.g/mL and 26.4 .mu.g/mL, respectively.
Rat studies have been published. The published data shows that
following a single dose of celgosivir in healthy rats at 35 mg/kg
PO, Cmax, Tmax and AUC values were 8.76.+-.1.15 .mu.g/ml,
0.44.+-.0.01 brand 10.5 respectively. (10) Oral bioavailability in
the rat was 93%, oral clearance (CL/F) was 2.5 L/h/kg, and
half-life was 2.7 hr. The anti-diarrhea agent loperamide had no
effect on the PK in either normal rats or those with castor
oil-induced diarrhea. (11)
In rats administered radiolabeled castanospermine, the majority of
radioactivity was detected within urine after PO (oral) or IV
dosing within 24 hr, and more than 92% was identified as
castanospermine. (12)
In rats with portal vein catheters administered 400 mg/kg
celgosivir PO, celgosivir concentrations in the portal vein were
approximately 4.4 to 13.2-fold higher than those in peripheral
circulation. The mean Cmax of celgosivir in the portal vein was 1.9
.mu.g/ml compared with 105.6 .mu.g/mL for castanospermine. AUC 0-60
min was 1.0 .mu.g/ml for celgosivir vs. 63.9 .mu.ghr/ml for
castanospermine. (10) Hence the majority of celgosivir was
converted to castanospermine prior to liver exposure, probably in
the gastrointestinal tract.
The maximum tolerated dose) in humans was determined in studies
conducted as part of HIV drug trials. The MTD was 400 mg per day
for 184 days. (13)
Example 8
Effect of Diet on Gastrointestinal Effects of Glucosidase
Inhibitors
Mice given a high dose of castanospermine (2 g/kg), when fed with
sucrose- and starch containing diets develop severe diarrhea and
high intestinal bacterial counts. (14) This is believed to be due
to off-target inhibition of gastrointestinal .alpha.-amylase,
sucrase, maltase and other enzymes involved in sucrose and glycogen
breakdown. Celgosivir is a weaker inhibitor than castanospermine of
the above-mentioned gastrointestinal enzymes. (10) When mice were
fed a diet of proteins, vitamins and glucose and which did not
contain sucrose or other complex sugars and carbohydrates, the GI
toxicity of castanospermine was ameliorated.
Example 9
Safety Pharmacology
Human Safety Pharmacology
Previous studies have provided valuable information on the clinical
pharmacology, pharmacokinetics, safety and tolerability of
celgosivir in humans. These studies have included single and
multiple dosing as well as QD dosing ranging from 10 to 600 mg for
2 to 12 weeks.
In humans, celgosivir has been shown to rapidly convert to
castanospermine where only the latter was detected in plasma. The
celgosivir was also shown to rapidly absorb with peak
concentrations of castanospermine occurring between 0.3 and 1.3 hr.
Cmax and AUC were also shown to increase in proportion to dose. The
terminal half-life for celgosivir in humans was 18 hr, and oral
clearance was 12 L/hr. (15)
Celgosivir is rapidly converted to castanospermine, which is
excreted unchanged in the urine, with no other metabolites
detected. Adverse events were largely gastrointestinal, namely
flatulence and diarrhea, of mild to moderate intensity.
Asymptomatic elevations in serum creatine kinase were also
observed, which were reversed within 2 weeks after discontinuation
of the drug. No serious adverse events have been reported.
Example 10
Treatment of a Dengue Infection in an Adult Subject with Celgosivir
Hydrochloride
##STR00006##
A human subject was orally administered an initial loading dose
o1400 mg of celgosivir followed by a maintenance dose of 200 mg
every 12 hr for a total of nine (9) doses. Drug was taken by the
human subject without food, i.e., 1 hr before consuming food or 2
hr afterwards. Subject was also placed on a special diet of
protein, vitamins, and glucose containing minimal complex sugars or
starches to minimize the likelihood of gastrointestinal side
effects. During treatment, several serum and urine samples were
obtained to analyze for drug levels.
On fifth day of treatment, after blood draws, administration of the
last dose, safety assessments, determination of risk for DHF or
DSS, and satisfactory clinical status, the human subject were
discharged. Blood sampling and safety assessments were then made on
Days 7, 10, and 15. Human subjects completed visual analog scales
for joint and muscle pain (VASP), and were evaluated for safety and
tolerability daily during treatment and at each subsequent visit.
Virological log reduction (VLR) was examined from Day 2 to 4. Viral
clearance, serum NS1 levels, leukocyte and platelet count, and,
hemoconcentration were also determined.
Example 11
Pharmacokinetic Model
Population pharmacokinetic (PK) analysis of castanospermine plasma
concentration data after administration of celgosivir was performed
using the non-linear mixed effects modelling software NONMEM
(version 7.2.0). (16) Pharmacokinetic parameters were estimated
using the first order conditional estimation method (FOCE) with
eta-epsilon interaction. Due to limited data during the input
phase, first-order absorption/formation of castanospermine was
assumed. Between-subject variability was assumed to be log-nomally
distributed. Model development was guided by objective function
value (OFV), precision of the parameter estimates, inspection of
standard goodness of lit plots and scientific plausability. The
emperical Bayes estimates from the final model were used to predict
individual concentrations for a standard saturated sampling design
(i.e., samples collected at 0, 0.25, 0.5, 1, 1.5, 2, 4, 6 and 12
hours post-dose)) after the first dose (400 mg) and dose 9 (day 5;
200 mg). These data were then use to determine the
non-compartmental PK analysis (NCA) parameters area under the
concentration-time curve over the dosing interval (AUC.sub.tau),
maximum concentration (C.sub.max), time of maximum concentration
(T.sub.max) and minimum concentration (C.sub.min). The NCA
parameter values were derived using the statistical analysis
software R (version 3.0.1). (17) NCA parameters after dose 9 were
only determined for subject who received this dose. The mean NCA PK
parameter values used to estimate the model-dependent parameter
values were taken from the results of a Phase I trial investigating
celgosivir and castanospermine PK after multiple doses (10-450
mg/day for 14 days) of celgosivir in HIV-positive patients. (18,
19)
These data are reproduced in Table 4.
TABLE-US-00004 TABLE 4 NCA PK Parameter Values Dose AUC.sub.ss
C.sub.max,ss t.sub.max C.sub.min,ss t.sub.1/2 (mg) (mg h/L) (ng/mL)
(h) (ng/mL) (h) 10 1.00 0.2266 0.7 8 13 20 1.75 0.52 1 11 27 40
3.61 1.07 1 23 30 80 6.53 1.72 0.3 29 16 160 13.30 3.73 0.8 70 21
240 21.50 5.76 0.6 110 16 360 32.10 6.319 0.7 179 15 450 39.80
10.50 NR 191 14 Note: The 80 and 450 mg dosing groups were excluded
from the analysis conducted. AUC.sub.ss is area under the
concentration-time curve at steady-state, C.sub.max,ss is the
maximum concentration at steady-state, t.sub.max is the time of
maximum concentration, C.sub.min is the minimum concentration at
steady-state, t.sub.1/2 is the terminal elimination half-life. NR
represents values that were not reported.
A sparse PK sampling design was employed with plasma samples
collected at pre-dose and 23, 25, 47, 49.5, 71, 74 and 95 hours
after first dose administration. (20) A total of 50 otherwise
healthy patients who had dengue fever were enrolled and randomly
assigned in a 1:1 ratio to receive either celgosivir or placebo.
Active treatment participants received a 400 mg loading dose
followed by 200 mg every 12 hours for 9 doses (i.e. 5 days of
treatment). A total of 163 concentrations from 24 individuals were
available for analysis. Pharmacokinetic data were best described by
a 1-compartment model with first-order input and elimination. No
time-dependent changes in PK were observed. A plot of measured
concentrations overlayed with the 10.sup.th to 90.sup.th prediction
interval from data simulated for 2000 individuals demonstrates the
model adequately describes the data and is illustrated in FIG. 7.
The predicted individual NCA paramer values after doses 1 and 9 are
summarised in Table 5. No major accumulation was observed, with
mean exposure (i.e. C.sub.max and AUC.sub.tau) on day 5 (dose 9;
200 mg) less than that after first dose (400 mg). Variability in
exposure between subjects was considered small to moderate.
TABLE-US-00005 TABLE 5 Predicted non-compartmental pharmacokinetic
parameters after doses 1 and 9. Dose 1 Dose 9 (400 mg; 0-12 h) (200
mg; 96-108 h) Parameter [n = 24] [n = 22] AUC.sub.tau Mean (SD)
48691 (2980) 25703 (1912) (ng h/mL) Median (Range) 48125 (42107,
54821) 25384 (21727, 28977) Cmax Mean (SD) 9703 (129) 5127 (135)
(ng/mL) Median (Range) 9684 (9394, 9950) 5105 (4844, 5422) Tmax
Mean (SD) 1 (na) 1 (na) (h) Median (Range) 1 (1, 1) 1 (1, 1) Cmin
Mean (SD) 684 (153) 357 (85) (ng/mL) Median (Range) 647 (385, 1025)
339 (198, 555)
Plots of simulated population mean castanospermine
concentration-time profiles after various celgosivir dosing
regimens are presented in FIG. 8A-H. The regimen that best produced
trough concentrations above 1000 ng/mL while reducing peak
concentrations relative to the dosing protocol described above (400
mg loading dose followed by 200 mg every 12 hours for 9 doses) was
administration of a 150 mg loading dose followed by 150 mg every 6
hours for 7 doses.
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The teachings of all patents, published applications and references
cited herein are incorporated by reference in their entirety.
While this invention has been particularly shown and described with
references to example embodiments thereof, it will be understood by
those skilled in the art that various changes in form and details
may be made therein without departing from the scope of the
invention encompassed by the appended claims.
* * * * *
References